Medium for detecting Staphylococcus aureus, sheet for detecting S. aureus comprising same, and method for detecting S. aureus using same

ABSTRACT

The purpose of the present invention is to provide a detection means whereby Staphylococcus aureus can be identified at a high accuracy. An aspect of the present invention relates to a medium for detecting S. aureus which comprises one or more kinds of nutrient components, a color developing agent capable of developing a color in the presence of α-glucosidase, a color developing agent capable of developing a color in the presence of phosphatase and 0.5 mg/cm3 or more of sodium colistin methanesulfonate. Another aspect of the present invention relates to a sheet for detecting S. aureus, said sheet comprising the aforesaid medium, and a method for detecting S. aureus with the use of the medium and sheet as described above.

TECHNICAL FIELD

The present invention relates to a medium used for detectingStaphylococcus aureus, a sheet used for detecting S. aureus comprisingsuch medium, and a method for detecting S. aureus using such medium andsuch sheet.

BACKGROUND ART

S. aureus is a type of bacteria that produces various toxins. Also, S.aureus is a pathogenic bacterium of septicemia, endocarditis, andserious bacterial infections, such as an infection of the lung orosteoarthritis. Accordingly, a means capable of detecting S. aureus in asimple manner with high accuracy has been desired at, for example,facilities that handle food or beverage products (e.g., factories andrestaurants) and medical institutions.

For example, Patent Literature 1 discloses a method of identifying andenumerating staphylococci in a sample containing one or more types ofbacteria, which is characterized by: the steps of i) inoculating aselective medium with an aliquot of a sample, wherein the mediumcomprises inhibitors to promote the growth of staphylococci, a firstsubstrate capable of producing an observable first color in the presenceof β-glucosidase, and second substrate capable of producing a secondcolor in the presence of staphylococci; ii) incubating the inoculatedmedium to produce the bacterial colony of sufficient size to allowvisualization of the bacterial colony in the presence of the first andsecond substrates in the medium; and iii) enumerating the colonyidentified by the presence of the second color of the second substrateto give the number of staphylococci in the sample. This literaturedescribes that the inhibitors can be selected from the group consistingof colistin methanesulfonate, nalidixic acid, and lithium chloride. Thisliterature also describes that the first substrate is anindolyl-glucopyranoside substrate providing a visual color change in thepresence of β-glucosidase and the second substrate provides a visualcolor change in the presence of staphylococcus.

Patent Literature 2 discloses a medium for the detection of S. aureusand/or coagulase-positive Staphylococci, which comprises a S. aureusculture medium and at least one enzyme substrate for demonstratingα-glucosidase activity. This literature describes an indoxyl-basedcompound as at least one enzyme substrate for demonstratingα-glucosidase activity.

In general, the medium and the culture medium disclosed in PatentLiteratures 1 and 2 are used in the form of a plane solid medium formedin a sterile petri dish with the use of a gelling agent such as agar. Insuch a case, occasionally, sophisticated techniques are required forpreparation of a plane solid medium, fractionation of samples, cultureof microorganisms, and other procedures.

Patent Literature 3 discloses a microorganism culture sheet having abase sheet, a culture layer formed on top of the base sheet, and a coversheet that covers the culture layer, wherein the culture layer ispattern-formed with a medium liquid comprising polyvinylpyrrolidone andat least one member selected from the group consisting of a gellingagent, a nutrient component, a color indicator, a selective agent, and asubstrate. This literature describes that cell count testing and thelike of food or beverage products can be performed in a simple andstable manner with the use of the microorganism culture sheet.

CITATION LIST Patent Literature

Patent Literature 1: JP H09-508279 A (1997)

Patent Literature 2: JP 2004-524041 A

Patent Literature 3: International Publication WO 2011/007802

SUMMARY OF INVENTION Technical Problem

To date, several means for detecting S. aureus have been known, althoughthese known means remain improvable. When the medium described in PatentLiterature 1 or 2 is applied to a particular embodiment, for example,staphylococcal bacteria other than S. aureus may be detected asfalse-positive strains.

Accordingly, the present invention is intended to provide a means ofdetection that enables identification of S. aureus with high accuracy.

Solution to Problem

The present inventors have examined the means described above in variousways. The present inventors added a color developing substrate specificto two types of enzymes expressed in S. aureus and a highly concentratedselection agent to a medium and found that S. aureus could be detectedwith high accuracy on the basis of the color of the colony formed on themedium as an indicator. The present invention has been completed on thebasis of such finding.

Specifically, the present invention is summarized as follows.

(1) A medium used for detecting Staphylococcus aureus comprising one ormore nutrient components, a color developer that develops color in thepresence of α-glucosidase, a color developer that develops color in thepresence of phosphatase, and colistin sodium methanesulfonate at 0.5mg/cm³ or more.(2) The medium according to (1), wherein the color developer thatdevelops color in the presence of α-glucosidase is6-chloro-3-indoxyl-α-D-glucoside, and the color developer that developscolor in the presence of phosphatase is 5-bromo-3-indoxyl phosphate.(3) The medium according to (1), wherein the color developer thatdevelops color in the presence of α-glucosidase is5-bromo-4-chloro-3-indoxyl-α-D-glucoside, and the color developer thatdevelops color in the presence of phosphatase is5-bromo-6-chloro-3-indoxyl phosphate.(4) The medium according to any of (1) to (3), which comprises colistinsodium methanesulfonate at 0.5 to 4.1 mg/cm³.(5) The medium according to (4), which comprises colistin sodiummethanesulfonate at 0.5 to 2.8 mg/cm³.(6) The medium according to (5), which comprises colistin sodiummethanesulfonate at 2.3 to 2.8 mg/cm³.(7) A microorganism culture substrate used for detection ofStaphylococcus aureus comprising a substrate and a culture layerprovided on an upper surface of the substrate, wherein the culture layercomprises the medium according to any of (1) to (6).(8) The microorganism culture substrate according to (7), which furthercomprises the substrate in the form of a sheet and a cover sheetcovering the culture layer, wherein the culture layer further comprisespolyvinylpyrrolidone and one or more gelling agents.(9) A method for detecting Staphylococcus aureus comprising:

a step of sample addition comprising adding a microorganism-containingsample to the medium according to any of (1) to (6) or the culture layerof the microorganism culture substrate according to (7) or (8);

a step of colony formation comprising incubating the medium ormicroorganism culture substrate added with the sample to form amicrobial colony; and

a step of strain identification comprising identifying Staphylococcusaureus on the basis of the color of the resulting microbial colony.

(10) The method according to (9), wherein the step of sample additioncomprises adding the microorganism-containing sample to the culturelayer of the microorganism culture substrate according to (7) or (8),and the culture layer of the microorganism culture substrate comprisescolistin sodium methanesulfonate at 0.2 mg/ml or more relative to thetotal volume of the microorganism-containing sample.

Advantageous Effects of Invention

The present invention can provide a means of detection that enablesidentification of S. aureus with high accuracy.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an embodiment of the microorganism culture substrate usedfor detecting S. aureus according to an aspect of the present invention;(a): a top view of the microorganism culture substrate according to anaspect of the present invention; (b): a schematic side cross sectionalview taken along I-I′ in (a); and (c): a schematic side cross sectionalview showing a culture layer covered with a folded cover sheet.

FIG. 2 shows another embodiment of the microorganism culture substrateused for detecting S. aureus according to an aspect of the presentinvention; (a): a top view of the microorganism culture substrateaccording to an aspect of the present invention; (b): a schematic sidecross sectional view taken along II-II′ in (a); and (c): a schematicside cross sectional view showing a culture layer covered with a foldedcover sheet.

FIG. 3 is a schematic side cross sectional view showing anotherembodiment of the microorganism culture substrate used for detection ofS. aureus according to an aspect of the present invention.

FIG. 4 is a schematic side cross sectional view showing anotherembodiment of the microorganism culture substrate used for detecting S.aureus according to an aspect of the present invention.

FIG. 5 is an external perspective view showing an embodiment of themicroorganism culture substrate used for detecting S. aureus accordingto an aspect of the present invention.

FIG. 6 is an external perspective view showing another embodiment of themicroorganism culture substrate used for detecting S. aureus accordingto an aspect of the present invention.

FIG. 7 shows photographs demonstrating the conditions of cell colonyformation after the color development in Experiment 5 and the colorsthereof; (a): the S. aureus ATCC25923 strain; (b): the S. aureusNBRC100910 strain; (c): the S. aureus NBRC12732 strain; (d): S.intermedius; (e): S. hyicus; and (f): S. epidermidis.

FIG. 8 shows photographs demonstrating the conditions of cell colonyformation after the color development in Experiment 5 and the colorsthereof; (a): S. saprophyticus subsp. saprophyticus; (b): S. xylosus;(c): S. sciuri; (d): Bacillus cereus; (e): B. licheniformis; and (f): B.subtilis.

FIG. 9 shows photographs demonstrating the conditions of cell colonyformation after the color development in Experiment 6 and the colorsthereof; (a): the S. aureus ATCC25923 strain; (b): the S. aureusNBRC100910 strain; (c): the S. aureus NBRC12732 strain; (d): S.intermedius; (e): S. hyicus; and (f): S. epidermidis.

FIG. 10 shows photographs demonstrating the conditions of cell colonyformation after the color development in Experiment 6 and the colorsthereof; (a): S. saprophyticus subsp. saprophyticus; (b): S. xylosus;(c): S. sciuri; (d): S. simulans; (e): S. haemolyticus; and (f): S.warneri.

FIG. 11 shows photographs demonstrating the conditions of cell colonyformation after the color development in Experiment 6 and the colorsthereof; (a): S. hominis; (b): S. cohnii; (c): S. capitis; (d): B.cereus; (e): B. licheniformis; and (f): B. subtilis.

DESCRIPTION OF EMBODIMENTS

The features of the present invention are described with reference toadequate figures herein. In the figures, dimensions and configurationsof components are exaggerated for clarification, so that actualdimensions and configurations are not accurately demonstrated.Accordingly, the technical scope of the present invention is not limitedto the dimensions and the configurations of the components demonstratedin the figures.

<1: Medium for Detecting Staphylococcus aureus>

An aspect of the present invention relates to a medium used fordetecting Staphylococcus aureus.

In the present invention, the term “Staphylococcus aureus” refers toStaphylococcus aureus. S. aureus is a Gram-positive bacterium of thegenus Staphylococcus. In general, the presence of S. aureus can beconfirmed by conducting a culture test using a S. aureus selectionmedium, such as a Baird-Parker agar medium or a mannitol salt agarmedium supplemented with egg yolk. A culture test involving the use ofsuch selection medium comprises allowing S. aureus to selectively growwith the aid of a selection agent added to the medium and identifying S.aureus on the basis of, for example, egg yolk reactivity or mannitoldegradability. A culture test involving the use of such selectionmedium, however, may occasionally detect staphylococcal bacteria otherthan S. aureus as false-positive strains. In general, accordingly, anadditional coagulase test using coagulase activity as an indicator isperformed after the culture test, so as to identify S. aureus. Coagulaseis an exoenzyme having activity of plasma clotting, which is expressedin S. aureus. Coagulase is known to be associated with humanpathogenicity caused by S. aureus. Accordingly, it was believed thatwith the use of coagulase activity as an indicator staphylococcalbacteria of interest can be detected separately from non-staphylococcalbacteria, such as Enterococcus and spore-forming Bacillus bacteria, andmany staphylococcal bacteria other than S. aureus. However, severalstaphylococcal bacteria other than S. aureus have thecoagulase-producing ability as with S. aureus. Therefore, suchstaphylococcal bacteria may be detected as false-positive strains in thetest for detecting S. aureus using coagulase activity as an indicator.

Accordingly, means for detecting S. aureus using activity of enzymesother than coagulase, such as various exoenzymes, includingα-glucosidase, phosphatase, β-glucuronidase, β-galactosidase, andβ-glucosidase, were developed (e.g., Patent Literature 1 and PatentLiterature 2). With such detection means, however, staphylococcalbacteria other than S. aureus may occasionally be detected asfalse-positive strains because of factors, such as a combination ofenzymes to be used together and/or culture conditions.

When α-glucosidase is used in combination with phosphatase, for example,it was considered that S. aureus would be expressed in response to boththe aforementioned enzymes as exoenzyme while staphylococcal bacteriaother than S. aureus would be expressed in response to either one orneither of the enzymes. Accordingly, it was considered that S. aureuscould be specifically detected with the use of activities of two typesof the enzymes, i.e., α-glucosidase and phosphatase, as indicators(Patent Literature 2). However, the present inventors have found thatseveral staphylococcal bacteria other than S. aureus could occasionallybe detected as false-positive strains by the test using activities oftwo types of the enzymes, α-glucosidase and phosphatase as indicators.

The present inventors have found that S. aureus could be detected withhigh accuracy on the basis of the color of the colony formed on a mediumas the indicators with the addition of a highly-concentrated selectionagent to the medium, in addition to two types of color developingsubstrates specific to exoenzymes other than coagulase expressed in S.aureus; i.e., α-glucosidase and phosphatase.

The medium used for detecting S. aureus according to the present aspectis required to comprise one or more nutrient components, a colordeveloper that develops color in the presence of α-glucosidase, a colordeveloper that develops color in the presence of phosphatase, andcolistin sodium methanesulfonate at high concentration. With the use ofthe medium according to the present aspect that comprises colistinsodium methanesulfonate at high concentration in addition to the twotypes of color developing substrates, S. aureus can be selectivelydetected with high accuracy without detecting staphylococcal bacteriaother than S. aureus as false-positive strains, as well asmicroorganisms other than those of staphylococcal bacteria.

One or more nutrient components to be used in the medium according tothe present aspect can be adequately selected from among substances thatare generally used in the art as nutrient components for media used fordetection of S. aureus. Examples of nutrient components include, but arenot limited to: peptone, tryptone, soytone, meat extract, yeast extract,sodium pyruvate, and an egg yolk mixture; sugar or sugar alcohol, suchas D(−)-mannitol; inorganic salts, such as potassium phosphate, sodiumphosphate, sodium carbonate, and potassium carbonate; and amino acids,such as glycine. A person skilled in the art can adequately determinethe concentration of the nutrient components within the range that isgenerally adopted in the art on the basis of, for example, the conditionof the medium according to the present aspect and/or the samplecontaining the target microorganisms at the time of use, the volumethereof, and/or the culture condition.

In the present invention, the term “selection agent” refers to acompound having antibacterial activity of suppressing the growth ofparticular types of microorganisms other than the target of detection;i.e., S. aureus. In this description, a compound having antibacterialactivity is occasionally referred to as an “antibacterial agent.” Themedium according to the present aspect comprises, as a selection agent,colistin sodium methanesulfonate at 0.5 mg/cm³ or more. Theconcentration of colistin sodium methanesulfonate is preferably 0.5 to4.1 mg/cm³, more preferably 0.5 to 2.8 mg/cm³, further preferably 2.3 to2.8 mg/cm³, and particularly preferably about 2.5 mg/cm³. Suchconcentration is defined as mass concentration relative to the totalvolume of the medium according to the present aspect. When an aspect ofthe present invention concerns a microorganism culture substrate usedfor detecting S. aureus comprising a substrate and a culture layercontaining the medium according to the present aspect provided on theupper surface of the substrate, for example, such concentration isdefined as mass concentration relative to the total volume of the driedculture layer of the medium according to the present aspect. In such acase, the mass concentration of colistin sodium methanesulfonaterelative to the total volume of a sample to be added to the driedculture layer is generally 0.2 mg/ml or more, preferably 0.2 to 1.6mg/ml, more preferably 0.2 to 1.1 mg/ml, further preferably 0.9 to 1.1mg/ml, and particularly preferably about 1 mg/ml. Colistin sodiummethanesulfonate is a compound known as an antibacterial agent againstGram-negative bacteria. It is generally used at a concentration of 0.1mg/ml (100 μg/ml) or lower, such as 1 to 20 μg/ml (Japanese Journal ofChemotherapy, Vol. 60, No. 4, pp. 446-467, 2012). In general, colistinsodium methanesulfonate is not used as an antibacterial agent againstGram-positive bacteria such as staphylococci. The present inventors havediscovered that colistin sodium methanesulfonate would exert the growthinhibitory activity at high concentration of 0.5 mg/cm³ or higher onstaphylococcal bacteria other than S. aureus (i.e., S. saprophyticussubsp. saprophyticus), 1.3 mg/cm³ or higher on S. carnosus, and 2.3mg/cm³ or higher on S. xylosus and S. sciuri. When the concentration ofcolistin sodium methanesulfonate is 0.5 mg/cm³ (0.2 mg/ml in terms ofmass concentration relative to the total volume of a sample added to thedried culture layer in the aspect of the microorganism culturesubstrate) or higher, accordingly, the growth of S. saprophyticus subsp.saprophyticus can be substantially suppressed, and detection of suchstrain as a false-positive strain can be prevented. When theconcentration of colistin sodium methanesulfonate is 2.3 mg/cm³ (0.9mg/ml in terms of mass concentration relative to the total volume of asample to be added to the dried culture layer in the aspect of themicroorganism culture substrate) or higher, in addition to S.saprophyticus subsp. saprophyticus, the growth of S. carnosus, S.xylosus, and S. sciuri is substantially suppressed, so that detection ofsuch strains as false-positive strains can be prevented. When theconcentration of colistin sodium methanesulfonate is 4.1 mg/cm³ (1.6mg/ml in terms of mass concentration relative to the total volume of asample to be added to the dried culture layer in the aspect of themicroorganism culture substrate) or less, the growth of major S. aureusstrains to be detected in the art is not inhibited, and such strains canbe detected as a positive strains. When the concentration of colistinsodium methanesulfonate is 2.8 mg/cm³ (1.1 mg/ml in terms of massconcentration relative to the total volume of a sample to be added tothe dried culture layer in the aspect of the microorganism culturesubstrate) or less, in particular, the growth of approximately all theS. aureus strains to be detected in the art is not inhibited, and suchstrains can be detected as positive strains. With the use of the mediumaccording to the present aspect containing colistin sodiummethanesulfonate at high concentration as described above, accordingly,detection of the staphylococcal bacteria other than S. aureus asfalse-positive strains can be substantially prevented.

The medium according to the present aspect can contain one or moreadditional selection agents, according to need. Such one or moreadditional selection agents can be adequately selected from amongcompounds having antibacterial activity that are commonly used in theart as selection agents to be added to a medium used for detecting S.aureus. Examples of one or more additional selection agents include, butare not limited to, lithium chloride, lithium sulfate, sodium chloride,sodium azide, potassium tellurite, nalidixic acid, deferoxamine,aztreonam, and bacitracin. Such one or more additional selection agentsare preferably a combination of lithium chloride, sodium azide, andnalidixic acid. A person skilled in the art can adequately determine theconcentration of one or more additional selection agents within therange that is generally adopted in the art on the basis of, for example,conditions of the medium according to the present aspect and/or a samplecontaining the target microorganisms at the time of use, the volumethereof, and/or the culture condition. The concentration of lithiumchloride is, for example, preferably 1 to 40 mg/cm³. The concentrationof sodium azide is, for example, preferably 20 to 200 μg/cm³. Theconcentration of nalidixic acid is, for example, preferably 10 to 40μg/cm³. Such concentrations are defined as mass concentration relativeto the total volume of the medium according to the present aspect. Whenan aspect of the present invention concerns a microorganism culturesubstrate used for detecting S. aureus, for example, such concentrationsare defined as mass concentration relative to the total volume of thedried culture layer comprising the medium according to the presentaspect. With the use of the medium according to the present aspectcomprising one or more additional selection agents, detection of thestaphylococcal bacteria other than S. aureus and other microorganisms asfalse-positive strains can be substantially prevented.

The concentration of a selection agent such as colistin sodiummethanesulfonate contained in the medium according to the present aspectcan be determined by, for example, purifying the selection agentcontained in a certain volume of the medium with one or more separationmeans selected from among, for example, solvent extraction,distillation, recrystallization, and chromatography such as adsorption,partition and gel filtration, and quantitatively analyzing the selectionagent with an analytic means, such as liquid chromatography/massanalysis (LC/MS).

The medium according to the present aspect contains a color developerthat develops color in the presence of α-glucosidase that is known as anexoenzyme of S. aureus. Examples of color developers that develop colorin the presence of α-glucosidase include, but are not limited to,6-chloro-3-indoxyl-α-D-glucoside (pink), 6-bromo-3-indoxyl-α-D-glucoside(red), 5-bromo-4-chloro-3-indoxyl-α-D-glucoside (light blue),5-bromo-4-chloro-3-indoxyl-N-methyl-α-D-glucoside (green),5-bromo-6-chloro-3-indoxyl-α-D-glucoside (magenta), and5-bromo-3-indoxyl-α-D-glucoside (blue). Such color developers are enzymesubstrates that develop color in the presence of α-glucosidase. Withreference to an embodiment in which the color developer is6-chloro-3-indoxyl-α-D-glucoside, for example, when the targetmicroorganism expresses α-glucosidase, 6-chloro-3-indoxyl-α-D-glucosidecontained in the medium is hydrolyzed to glucose and an indole compound(6-chloro-3-indole) as the microorganism grows. The resulting indolecompound is further dimerized, and the resultant turns pink. Other colordevelopers are also hydrolyzed and dimerized, and the colors indicatedin the parentheses are then developed.

The medium according to the present aspect contains a color developerthat develops color in the presence of phosphatase that is known as anexoenzyme of S. aureus. Examples of color developers that develop colorin the presence of phosphatase include, but are not limited to,5-bromo-3-indoxyl phosphate (blue), 6-chloro-3-indoxyl phosphate (pink),6-bromo-3-indoxyl phosphate (red), 5-bromo-4-chloro-3-indoxyl phosphate(light blue), and 5-bromo-6-chloro-3-indoxyl phosphate (magenta). Suchcolor developers are enzyme substrates that develop color in thepresence of phosphatase. With reference to an embodiment in which thecolor developer is 5-bromo-3-indoxyl phosphate, for example, when thetarget microorganism expresses phosphatase, 5-bromo-3-indoxyl phosphatecontained in the medium is hydrolyzed to phosphoric acid and an indolecompound (5-bromo-3-indole) as the microorganism grows. The resultingindole compound is further dimerized, and the resultant turns blue todark blue. Other color developers are also hydrolyzed and dimerized, andthe color indicated in the parentheses are then developed.

The medium according to the present aspect comprises a color developerthat develops color in the presence of α-glucosidase and a colordeveloper that develops color in the presence of phosphatase. The colordeveloper that develops color in the presence of α-glucosidase ispreferably 6-chloro-3-indoxyl-α-D-glucoside, and the color developerthat develops color in the presence of phosphatase is preferably5-bromo-3-indoxyl phosphate. Alternatively, the color developer thatdevelops color in the presence of α-glucosidase is preferably5-bromo-4-chloro-3-indoxyl-α-D-glucoside, and the color developer thatdevelops color in the presence of phosphatase is preferably5-bromo-6-chloro-3-indoxyl phosphate. S. aureus has the ability toproduce α-glucosidase and the ability to produce phosphatase. When S.aureus is present in the medium according to the present aspect inwhich, for example, the color developer that develops color in thepresence of α-glucosidase is 6-chloro-3-indoxyl-α-D-glucoside and thecolor developer that develops color in the presence of phosphatase is5-bromo-3-indoxyl phosphate, accordingly, the colony formed by suchstrain was deduced to turn, for example, purple, which is a mixed colorof pink developed by α-glucosidase activity and blue to dark bluedeveloped by phosphatase activity. When S. aureus was present in themedium according to the present aspect containing the color developersin combination as described, surprisingly, it has been found that thestrain grew and the resulting colony turned blue to dark blue. When S.intermedius having both the ability to produce α-glucosidase and theability to produce phosphatase among staphylococcal bacteria other thanS. aureus is present in the medium according to the present aspectcontaining the color developers in combination as described, incontrast, the strain grows and the resulting colony turns purple. When,among staphylococcal bacteria other than S. aureus, staphylococcalbacteria other than S. intermedius, such as S. saprophyticus subsp.saprophyticus, S. carnosus, S. xylosus, or S. sciuri, are present in themedium according to the present aspect containing the color developersin combination as described, the growth of the strain is inhibited bythe highly concentrated colistin sodium methanesulfonate. Thus, thestrain cannot exert the ability to produce α-glucosidase and/or theability to produce phosphatase, and no color is developed. When Bacillusbacteria other than staphylococci are present in the medium according tothe present aspect containing the color developers in combination asdescribed, in addition, the growth of the strain is inhibited.Alternatively, the strain occasionally grows, and the resulting colonyturns pink. When S. aureus is present in the medium according to thepresent aspect in which a color developer that develops color in thepresence of α-glucosidase is 5-bromo-4-chloro-3-indoxyl-α-D-glucoside,and a color developer that develops color in the presence of phosphataseis 5-bromo-6-chloro-3-indoxyl phosphate, it has been found that thestrain would grow and the resulting colony would turn magenta. When,among staphylococcal bacteria other than S. aureus, S. intermediushaving both the ability to produce α-glucosidase and the ability toproduce phosphatase is present in the medium according to the presentaspect containing the color developers in combination described above,in contrast, the strain grows and the resulting colony turns gray. When,among staphylococcal bacteria other than S. aureus, staphylococcalbacteria other than S. intermedius, such as S. saprophyticus subsp.saprophyticus, S. xylosus, or S. sciuri, are present in the mediumaccording to the present aspect containing the color developers incombination described above, the growth of the strain is inhibited bythe highly concentrated colistin sodium methanesulfonate. Thus, thestrain cannot exert the ability to produce α-glucosidase and/or theability to produce phosphatase, and no color is developed. When Bacillusbacteria other than staphylococci are present in the medium according tothe present aspect containing the color developers in combinationdescribed above, the growth of the strain is inhibited, or the strainoccasionally grows and the resulting colony turns gray or blue. With theuse of the medium according to the present aspect containing colistinsodium methanesulfonate at high concentration in addition to the twotypes of color developers, accordingly, S. aureus can be selectivelydetected with high accuracy without detecting staphylococcal bacteriaother than S. aureus and other microorganisms as false-positive strains.

The medium according to the present aspect can contain at least onesolvent, according to need. Examples of at least one solvent include,but are not limited to, water, a lower alcohol (e.g., an alcohol having1 to 6 carbon atoms such as methanol, ethanol, or 2-propanol (isopropylalcohol)), a higher alcohol (e.g., an alcohol having 7 or more carbonatoms such as 1-heptanol or 1-octanol), and dimethyl sulfoxide (DMSO).At least one solvent is preferably water. With the use of the solvent,which is preferably water, the medium according to the present aspectcontaining the solvent enables detection of the target of detection(i.e., S. aureus) with high accuracy while refraining from inhibitingthe growth thereof.

The medium according to the present aspect can be used in the form ofeither a solid medium or liquid medium. When the medium according to thepresent aspect is a liquid medium, it can be used for liquid culturewhile it is accommodated in a culture vessel that is generally used inthe art, such as a flask, petri dish, tube, or multi-well plate.

When the medium according to the present aspect is a solid medium, itcan be used in any form. In this embodiment, for example, the mediumaccording to the present aspect can be used in the form of a plane solidmedium accommodated in a culture vessel that is generally used in theart, such as a petri dish or multi-well plate, or in the form of aculture substrate in which the medium is provided on an upper surface ofthe substrate in a desired form. In the embodiment of a solid medium, ingeneral, the medium according to the present aspect contains one or moregelling agents. One or more gelling agents can be adequately selectedfrom among compounds that are generally used in the art as gellingagents that solidify a medium used for detecting S. aureus. Examples ofone or more gelling agents include, but are not limited to,carragheenan, xanthan gum, Locust bean gum, psyllium seed gum, guar gum,hydroxyethyl cellulose, carboxymethyl cellulose, alginic acid, alginate,agar, and gelatin. One or more gelling agents are preferably acombination of psyllium seed gum and guar gum. A person skilled in theart can adequately determine the concentration of one or more gellingagents within the range that is generally adopted in the art on thebasis of, for example, conditions of the medium according to the presentaspect and/or a sample containing the target microorganisms at the timeof use, volumes thereof, and/or culture conditions.

The medium according to the present aspect with the features describedabove enables selective detection of S. aureus with high accuracywithout detecting coexisting staphylococci and other microorganisms asfalse-positive strains.

<2: Microorganism Culture Substrate Used for Detecting Staphylococcusaureus>

The medium according to an aspect of the present invention can be usedas a solid medium of any configuration. Accordingly, another aspect ofthe present invention relates to a microorganism culture substrate usedfor detecting S. aureus, which comprises a substrate and a culture layerprovided on an upper surface of the substrate comprising the mediumaccording to the present invention.

According to the present aspect, the configuration of the substrate canbe adequately selected from among various configurations that aregenerally employed in the art, such as a sheet, film, plate, or vessel(e.g., a circular or square petri dish).

According to the present aspect, the configuration of the culture layeris not particularly limited, and it can be adequately selected inaccordance with, for example, the configuration of the substrate.According to the present aspect, the culture layer comprises the mediumaccording to an aspect of the present invention. The medium according toan aspect of the present invention contained in the culture layer of themicroorganism culture substrate according to the present aspect has thefeatures described above. Accordingly, the microorganism culturesubstrate according to the present aspect enables selective detection ofS. aureus with high accuracy without detecting coexisting staphylococciand other microorganisms as false-positive strains.

The microorganism culture substrate according to the present aspectpreferably has a sheet-formed substrate (hereafter, it is also referredto as a “substrate sheet”). The microorganism culture substrateaccording to the present aspect having a substrate sheet (hereafter, itis also referred to as a “microorganism culture sheet used for detectingS. aureus” or simply as a “microorganism culture sheet”) can comprise asubstrate sheet, a culture layer provided on an upper surface of thesubstrate sheet, and a cover sheet covering the culture layer. In such acase, the culture layer comprises, for example, the medium according toan aspect of the present invention, one or more fixing agents, and oneor more gelling agents.

A microorganism culture sheet having a substrate sheet and a culturelayer containing a solid medium provided on an upper surface of thesubstrate sheet is known in the art. Such microorganism culture sheet isdisclosed in, for example, International Publication WO 2011/007802 andJP 2014-90701 A. Any configuration of a microorganism culture sheetdisclosed in the above literature and known in the art can be adoptedfor the microorganism culture substrate used for detecting S. aureusaccording to the present aspect.

FIG. 1 shows an embodiment of a microorganism culture substrate used fordetecting S. aureus according to the present aspect. In FIG. 1, (a)shows a top view of the microorganism culture substrate according to thepresent aspect; (b) shows a schematic side cross sectional view takenalong I-I′ in (a); and (c) shows a schematic side cross sectional viewshowing a culture layer covered with a folded cover sheet. Themicroorganism culture sheet having a sheet-formed substrate, which is anembodiment of the microorganism culture substrate according to thepresent aspect, comprises: a substrate sheet 10; a culture layer 30provided on an upper surface of the substrate sheet 10; and a coversheet 40 covering the culture layer 30, as shown in, for example,FIG. 1. The substrate sheet 10 may be a single-layered sheet or amulti-layered sheet composed of a laminate of a plurality of sheets madeof the same or different materials. Also, the cover sheet 40 may be asingle-layered sheet or a multi-layered sheet composed of a laminate ofa plurality of sheets made of the same or different materials. Further,the culture layer 30 may be of a single-layered structure or amulti-layered structure composed of a laminate of a plurality of layershaving the same or different compositions. When the culture layer 30 isof a multi-layered structure, it is sufficient if at least one layercontains the medium according to the present aspect.

The configuration of the substrate sheet and that of the cover sheet ofthe microorganism culture sheet according to the present embodiment arenot particularly limited. For example, the configuration of thesubstrate sheet 10 and that of the cover sheet 40 may be a square,another polygon such as a triangle, a circle such as a true or ovalcircle, or irregular. The configuration of the substrate sheet 10 may bethe same with or different from that of the cover sheet 40. The size ofthe substrate sheet 10 may be the same with or different from that ofthe cover sheet 40. The configuration and the size of the substratesheet 10 are preferably the same with those of the cover sheet 40. Insuch a case, the cover sheet 40 covers the entire upper surface of thesubstrate sheet 10 on which the culture layer 30 is provided, so thatthe culture layer 30 can be protected.

The configuration of the culture layer of the microorganism culturesheet according to the present embodiment is not particularly limited.For example, the configuration of the culture layer 30 may be a circlesuch as a true or oval circle, a polygon, such as a square (i.e., a trueor oblong square) or triangle, or irregular, as shown in FIG. 1. Thesize of the culture layer 30 can be adequately determined, as long as itcan be provided on an upper surface of the substrate sheet 10. Theculture layer 30 is preferably a circle. In such a case, the sample maybe added to an area in the vicinity of the center of the culture layer30, so that the sample can be uniformly dispersed throughout the culturelayer 30.

In the microorganism culture sheet according to the present embodiment,the cover sheet is preferably fixed to the substrate sheet. A means forfixing the cover sheet is not particularly limited. For example, thecover sheet 40 can be directly fixed onto the substrate sheet 10 by anymeans such as pressure bonding, as shown in FIGS. 1 and 2.Alternatively, the cover sheet 40 can be fixed to the substrate sheet 10with the aid of a fixing member 25, such as a double-sided tape oradhesive, as shown in FIGS. 3 and 4.

The microorganism culture substrate used for detecting S. aureusaccording to the present aspect can have a frame layer provided tosurround the outer periphery of the culture layer, according to need.FIG. 2 shows another embodiment of the microorganism culture substrateused for detecting S. aureus according to the present aspect. In FIG. 2,(a) shows a top view of the microorganism culture substrate according tothe present aspect; (b) shows a schematic side cross sectional viewtaken along II-II′ in (a); and (c) shows a schematic side crosssectional view showing a culture layer covered with a folded coversheet. The microorganism culture sheet having a sheet-formed substrate,which is an embodiment of the microorganism culture substrate accordingto the present aspect, comprises: a substrate sheet 10; a culture layer30 provided on an upper surface of the substrate sheet 10; a frame layer20 provided to surround the culture layer 30; and a cover sheet 40covering the culture layer 30, as shown in, for example, FIG. 2. Whenthe microorganism culture sheet has the frame layer 20, the area inwhich a sample is allowed to spread can be limited to the area withinthe culture layer 30 at the time of the addition of the sample to theculture layer 30. The frame layer 20 may be provided to be in closecontact with or distant from the outer periphery of the culture layer30. The frame layer 20 is preferably provided to be in close contactwith the outer periphery of the culture layer 30. Thus, the area inwhich a sample is allowed to spread can be definitely limited to thearea within the culture layer 30 at the time of the addition of thesample to the culture layer 30. The configuration of the frame layer 20is not particularly limited, and a person skilled in the art can selectan adequate configuration in accordance with the configuration of theculture layer 30. For example, the configuration of the frame layer 20may be a circle such as a true or oval circle, a polygon, such as asquare (i.e., a true or oblong square) or triangle, or irregular, asshown in FIG. 2. The frame layer 20 is preferably a circle, and it ismore preferably a circle and provided to be in close contact with theouter periphery of the culture layer 30. With the addition of a samplein an area in the vicinity of the center of the culture layer 30, thesample is allowed to uniformly spread over the entire area of theculture layer 30, and the area in which the sample is allowed to spreadcan be definitely limited to the area within the culture layer 30.

When the microorganism culture substrate used for detecting S. aureusaccording to the present aspect comprises the culture layer and theframe layer (in a case where it is present), at least one culture layerand at least one frame layer may be provided on an upper surface of thesubstrate. Specifically, a plurality of culture layers and frame layers(in a case where they are present) may be provided on an upper surfaceof the substrate. In such embodiment, a plurality of culture layers andframe layers (in a case where they are present) can be provided at anypositions on an upper surface of the substrate.

A person skilled in the art can adequately determine the size of themicroorganism culture substrate used for detecting S. aureus accordingto the present aspect in accordance with, for example, the volume of thetarget microorganism-containing sample. In the embodiment of amicroorganism culture sheet having a sheet-formed substrate in which theconfiguration of the substrate sheet 10 is square, for example, a lengthof a side of the substrate sheet 10 is generally 50 to 100 mm, andtypically 70 to 90 mm. A thickness thereof is generally 25 to 1500 μm,and typically 50 to 500 μm. The configuration and the size of the coversheet 40 are preferably the same with those of the substrate sheet 10. Athickness of the cover sheet 40 is preferably 10 to 200 μm, and morepreferably 20 to 70 μm. When the configuration of the culture layer 30is circular, a diameter of the culture layer 30 is preferably 20 to 80mm, and more preferably 30 to 70 mm. When the configuration of theculture layer 30 is not circular, a diameter of the maximal circleinscribed in the figure defined by the outer periphery of the culturelayer 30 is preferably within the range described above. A thickness ofthe culture layer 30 is preferably 150 to 250 μm, and more preferably190 to 210 μm. A thickness of the culture layer 30 is defined as anaverage distance from the lower surface of the dried culture layer 30(i.e., the surface that is in contact with the substrate sheet 10) tothe upper surface of the culture layer 30 of the microorganism culturesheet according to the present embodiment. A thickness of the culturelayer 30 can be determined by measuring, for example, the distancebetween the lower surface and the upper surface of the substrate sheet10 and the distance between the lower surface of the substrate sheet 10and the upper surface of the dried culture layer 30 at several positionsand calculating the average of differences in both distances. When thesize of the culture layer 30 is within the range described above, agiven amount of a sample can be absorbed and retained with certainty.

When the microorganism culture substrate used for detecting S. aureusaccording to the present aspect comprises a frame layer, a personskilled in the art can adequately determine the size of the frame layerin accordance with the size of the culture layer. In the embodiment of amicroorganism culture sheet having a sheet-formed substrate, forexample, a height of the frame layer 20 is preferably higher than theheight of the culture layer 30 by 100 to 1200 μm, more preferably by 200to 1000 μm, and further preferably by 300 to 800 μm. A width of theframe layer 20 is preferably 0.5 to 5.0 mm, and more preferably 1.0 to3.0 mm. A height of the frame layer 20 is defined as an average distancefrom the lower surface of the frame layer 20 (i.e., the surface that isin contact with the substrate sheet 10) to the upper surface of theframe layer 20 of the microorganism culture sheet according to thepresent embodiment. A height of the frame layer 20 can be determined bymeasuring, for example, the distance between the lower surface and theupper surface of the substrate sheet 10 and the distance between thelower surface of the substrate sheet 10 and the upper surface of theframe layer 20 at several positions and calculating the average ofdifferences in both distances. A width of the frame layer 20 is definedas an average distance between the side surfaces of the frame layer 20.When the size of the frame layer 20 is within the range described above,effusion of a sample occurring when the sample is added to the culturelayer 30 can be prevented. When the culture layer 30 is covered with thecover sheet 40, generation of gaps between the cover sheet 40 and theculture layer 30 can be substantially prevented. Thus, a sample isallowed to spread in the culture layer 30 with certainty.

A substrate of the microorganism culture substrate according to thepresent aspect preferably comprises, as a main component, at least oneplastic material selected from the group consisting of polyester,polyethylene, polypropylene, polystyrene, polycarbonate, and polyvinylchloride. The substrate comprising the plastic material as a maincomponent is water resistant and solvent resistant. Thus, a culturelayer comprising water and/or a solvent can be provided on the uppersurface of the substrate. Further, the substrate comprising the plasticmaterial as a main component is heat resistant and it has printability.Thus, a culture layer can be provided on the upper surface of thesubstrate by means of, for example, printing, coating, or spraying.Furthermore, the substrate comprising the plastic material as a maincomponent has excellent transparency. Thus, a microbial colony grown inthe culture layer can be observed through the light penetrating thesubstrate. Alternatively, the substrate may be colored with the additionof a coloring agent or foaming agent to the plastic material or viaother means and used in the colored state. With the use of the coloredsubstrate, the color of the colony grown in the culture layer can beevaluated more apparently.

According to an embodiment of a microorganism culture sheet having asheet-formed substrate in which a substrate sheet has a multi-layeredstructure, such substrate sheet may be a laminate of the sheetsidentical to each other mainly composed of the plastic materialdescribed above or a laminate of sheets different from each other.Alternatively, a substrate sheet of a multi-layered structure may be alaminate of sheets composed mainly of paper and/or synthetic resin andsheets composed mainly of the plastic material described above differentfrom or identical to each other. Examples of substrate sheets of amulti-layered structure composed mainly of paper and synthetic resininclude YUPO® (Yupo Corporation) and CRISPR® (Toyobo Co., Ltd.).

In the microorganism culture sheet according to the embodiment of thepresent invention, a cover sheet is employed for the purpose ofprevention of contamination of the culture layer and/or prevention ofmoisture evaporation from the culture layer. The cover sheet preferablycomprises, as a main component, at least one plastic material selectedfrom the group consisting of polyester, polyethylene, polypropylene,polystyrene, polycarbonate, and polyvinyl chloride. The cover sheetcomposed mainly of the plastic material has excellent transparency.Thus, a microbial colony grown in the culture layer can be observedthrough the light penetrating the cover sheet. The cover sheet 40 mayhave a squared pattern of a given size as an indicator of observation asshown in, for example, FIGS. 5 and 6. According to the presentembodiment, a squared pattern can be formed by printing the pattern onthe cover sheet surface using an ink that is insoluble in water and doesnot substantially influence the microbial growth.

The culture layer of the microorganism culture substrate according tothe present aspect can comprise one or more fixing agents. In thepresent invention, the term “fixing agent” refers to a compound thatmodifies the viscosity of the medium contained in the culture layer andbrings the culture layer into close contact with the surface of thesubstrate sheet. According to an embodiment of a microorganism culturesheet comprising a sheet-formed substrate, one or more fixing agents arepreferably polyvinylpyrrolidone. With the use of polyvinylpyrrolidone asone or more fixing agents, the medium components contained in theculture layer can be uniformly dispersed. By increasing the viscosity ofthe medium according to an aspect of the present invention, also, aculture layer of the size of interest can be provided at a position ofinterest on the upper surface of the substrate sheet when producing themicroorganism culture sheet according to the present embodiment. Inaddition, polyvinylpyrrolidone is highly adhesive to the substrate sheetcomposed mainly of the plastic material. Thus, the culture layer can beadhered to the surface of the substrate sheet without the use of anadhesive agent or the like. The concentration of polyvinylpyrrolidoneused as one or more fixing agents is preferably 15 to 20%. Suchconcentration is defined as the final concentration (mass percentage)relative to the total mass of the dried culture layer in themicroorganism culture sheet according to the present embodiment. Byincorporating polyvinylpyrrolidone as one or more fixing agents, aculture layer with desired properties can be prepared.

The culture layer of the microorganism culture substrate according tothe present aspect can comprise one or more gelling agents. As one ormore gelling agents, gelling agents exemplified above as components ofthe medium according to an aspect of the present invention can be used.According to an embodiment of the microorganism culture sheet having asheet-formed substrate, one or more gelling agents are preferably acombination of psyllium seed gum and guar gum. The concentration of oneor more gelling agents is preferably 50 to 60%. Such concentration isdefined as the final concentration (mass percentage) relative to thetotal mass of the dried culture layer in the microorganism culture sheetaccording to the present embodiment. By incorporating one or moregelling agents, a culture layer with desired properties can be prepared.

The culture layer of the microorganism culture substrate according tothe present aspect comprises one or more plasticizers, according toneed. Examples of one or more plasticizers include glycerin, a glycerinderivative-based plasticizer, and polyethylene glycol. According to anembodiment of a microorganism culture sheet having a sheet-formedsubstrate, one or more plasticizers are preferably glycerin. Theconcentration of one or more plasticizers is preferably 5 to 10%. Suchconcentration is defined as the final concentration (mass percentage)relative to the total mass of the dried culture layer in themicroorganism culture sheet according to the present embodiment. Byincorporating one or more plasticizers, plasticity, flexibility,elasticity, and other properties of the culture layer can be improved.

The microorganism culture sheet having a sheet-formed substrateaccording to the present aspect can be produced on the basis of anymethod for producing a microorganism culture sheet known in the art,which is disclosed in, for example, WO 2011/007802 or JP 2014-90701 A.The microorganism culture sheet according to the present embodiment canbe produced by, for example, a method comprising a step of preparing asubstrate sheet, a step of pattern forming a culture layer on an uppersurface of the substrate sheet, and a step of fixing the substrate sheetand the cover sheet. When the microorganism culture sheet according tothe present embodiment has a frame layer, it is preferable that themethod of production described above further comprise a step of forminga frame layer on an upper surface of the substrate sheet.

In the method for producing the microorganism culture sheet according tothe present embodiment, a step of pattern forming a culture layer on anupper surface of the substrate sheet further comprises: a step ofpreparing a medium liquid comprising the components of the culture layersuspended in one or more solvents; a step of applying the medium liquidto an upper surface of the substrate sheet; and a step of drying theapplied medium liquid to form a culture layer with a givenconfiguration. When preparing the medium liquid, one or more solventsused for viscosity modification or other purposes can be adequatelyselected from among the solvents exemplified above as components of themedium according to an aspect of the present invention. As one or moresolvents used for medium liquid preparation, a lower alcohol ispreferable, methanol, ethanol, or 2-propanol (isopropyl alcohol) is morepreferable, and methanol is further preferable. In general, the mediumliquid contains all the components contained in the culture layer in thestep of preparation; however, it is not necessary for the medium liquidto contain some components contained in the culture layer in the step ofpreparation. In such a case, during the step of applying the mediumliquid to the upper surface of the substrate sheet or at any time afterthe step, the remaining components may be added to the applied mediumliquid or the formed culture layer.

The step of applying a medium liquid to an upper surface of thesubstrate sheet can be performed by means of, for example, printing,coating, or spraying. In the subsequent step of drying the appliedmedium liquid, a solvent contained in the medium liquid may be removedby evaporation to form a culture layer of a given configuration.Polyvinylpyrrolidone contained in the culture layer is highly adhesiveto the substrate sheet composed mainly of the plastic material describedabove. By performing the step of applying a medium liquid to an uppersurface of the substrate sheet and the subsequent step of drying theapplied medium liquid, accordingly, the culture layer can be adhered tothe surface of the substrate sheet without the use of an adhesive agentor the like.

The method for producing the microorganism culture sheet according tothe present embodiment may further comprise a step of sterilizing theobtained microorganism culture sheet. Examples of means for sterilizingthe microorganism culture sheet include sterilization via application ofradioactive rays such as gamma rays and chemical sterilization with theuse of ethylene oxide gas.

By performing the method comprising the steps described above, themicroorganism culture sheet having a sheet-formed substrate can beproduced.

<3: Method for Detecting Staphylococcus aureus>

The medium and the microorganism culture substrate according to anaspect of the present invention can be used for selectively detecting S.aureus with high accuracy from a sample containing many microorganismsincluding staphylococcal bacteria other than S. aureus. Accordingly,another aspect of the present invention relates to a method fordetecting S. aureus with the use of the medium and the microorganismculture substrate according to an aspect of the present invention. Themethod for detecting S. aureus according to the present aspect comprisesa step of sample addition, a step of colony formation, and a step ofstrain identification.

[3-1: Step of Sample Addition]

This step comprises adding a microorganism-containing sample to themedium and the culture layer of the microorganism culture substrateaccording to an aspect of the present invention.

A sample used in this step generally contains S. aureus, and the sampleoccasionally contains S. aureus and further microorganisms other than S.aureus. As described above, the medium and the microorganism culturesubstrate according to an aspect of the present invention enableselective detection of S. aureus with high accuracy from a samplecontaining further microorganisms such as staphylococcal bacteria otherthan S. aureus. Accordingly, the further microorganisms contained in thesample are not particularly limited. Examples of the furthermicroorganisms contained in the sample include, but are not limited to,bacteria of non-staphylococcal bacteria, such as Escherichia coli,Enterococcus, and Bacillus bacteria, in addition to staphylococcalbacteria other than S. aureus. By the method according to the presentaspect, S. aureus can be selectively detected with high accuracy fromsamples containing many microorganisms exemplified above.

A microorganism-containing sample can be prepared from any target, suchas a product (e.g., a food, beverage, medical, or animal feed product),equipment, apparatus, and place in which S. aureus may be present.

In this step, a microorganism-containing sample is generally used in theform of liquid. In such a case, a solvent of the sample is generallysterile water. A solvent can contain an inorganic salt, buffer, medium,and other components, according to need.

In this step, a person skilled in the art can adequately determine thevolume of a sample to be added to the medium and the culture layer ofthe microorganism culture substrate according to an aspect of thepresent invention in accordance with, for example, the size of themedium and the culture layer of the microorganism culture substrateaccording to an aspect of the present invention. In the embodiment ofthe microorganism culture substrate according to an aspect of thepresent invention having the sheet-formed substrate as shown in FIG. 1;i.e., the microorganism culture sheet, for example, about 1 ml of asample is preferably added. With the addition of a sample in the volumedescribed above, the added sample can be absorbed and retained by theculture layer with certainty.

When the microorganism culture substrate according to an aspect of thepresent invention, for example, the microorganism culture sheet, is usedin this step, the culture layer of the microorganism culture substratepreferably comprises colistin sodium methanesulfonate at 0.2 mg/ml ormore relative to the total volume of the microorganism-containingsample. In such a case, the concentration of colistin sodiummethanesulfonate contained in the culture layer of the microorganismculture substrate is preferably 0.2 to 1.6 mg/ml, more preferably 0.2 to1.1 mg/ml, further preferably 0.9 to 1.1 mg/ml, and particularlypreferably about 1 mg/ml. With the use of the microorganism culturesubstrate containing colistin sodium methanesulfonate at the highconcentration as described above, detection of the staphylococcalbacteria other than S. aureus as false-positive strains can besubstantially prevented.

In the method according to the present aspect, S. aureus is identifiedon the basis of the color of the colony formed in the medium and theculture layer of the microorganism culture substrate according to anaspect of the present invention. When the cell density of themicroorganisms contained in the sample is high, accordingly, it may bedifficult to separate and identify the resulting colonies as a pluralityof single colonies. It is thus preferable to dilute themicroorganism-containing sample with a solvent in advance to adjust thecell density to an adequate level. A person skilled in the art canadequately determine the cell density of the microorganisms contained ina sample in accordance with the volume of the sample added and the sizeof the medium used and the culture layer of the microorganism culturesubstrate according to an aspect of the present invention. In theembodiment of the microorganism culture substrate according to an aspectof the present invention having a sheet-formed substrate as shown inFIG. 1; i.e., the microorganism culture sheet, for example, the celldensity of the microorganisms contained in a sample is preferably 1 to500 cfu/ml. When the cell density of the microorganisms contained in asample is within the range described above, all the microorganismscontained in the sample can be separated and identified as a singlecolony in the culture layer with the addition of about 1 ml of thesample to the culture layer.

[3-2: Step of Colony Formation]

This step comprises incubating the medium or microorganism culturesubstrate supplemented with a sample to form microbial colonies.

A person skilled in the art can adequately determine conditions forincubation of the medium or microorganism culture substrate supplementedwith a sample in accordance with the conditions for culture ofmicroorganisms such as S. aureus generally employed in the art.

[3-3: Step of Strain Identification]

This step comprises identifying S. aureus on the basis of the color ofthe formed microbial colony.

When S. aureus is present in the medium or the culture layer of themicroorganism culture substrate according to an aspect of the presentinvention in which, for example, a color developer that develops colorin the presence of α-glucosidase is 6-chloro-3-indoxyl-α-D-glucoside anda color developer that develops color in the presence of phosphatase is5-bromo-3-indoxyl phosphate, as described above, the colony formed bysuch strain turns blue to dark blue. When, among staphylococcal bacteriaother than S. aureus, S. intermedius having both the ability to produceα-glucosidase and the ability to produce phosphatase is present in themedium or the culture layer of the microorganism culture substrateaccording to an aspect of the present invention that contains the colordevelopers in combination described above, in contrast, the colonyformed by such strain turns purple. When staphylococcal bacteria otherthan S. intermedius, such as S. saprophyticus subsp. saprophyticus, S.carnosus, S. xylosus, or S. sciuri among staphylococcal bacteria otherthan S. aureus, are present in the medium or the culture layer of themicroorganism culture substrate according to an aspect of the presentinvention that contains the color developers in combination describedabove, for example, the growth of the strain is inhibited by the highlyconcentrated colistin sodium methanesulfonate, the strain cannot exertthe ability to produce α-glucosidase and/or the ability to producephosphatase, and the color is not developed. When bacteria of Bacillusother than staphylococcus are present in the medium or the culture layerof the microorganism culture substrate according to an aspect of thepresent invention that contains the color developers in combinationdescribed above, in addition, the growth of the strain is inhibited.Alternatively, the strain may grow and the resulting colony may turnpink. When S. aureus is present in the medium or the culture layer ofthe microorganism culture substrate according to an aspect of thepresent invention in which a color developer that develops color in thepresence of α-glucosidase is 5-bromo-4-chloro-3-indoxyl-α-D-glucosideand a color developer that develops color in the presence of phosphataseis 5-bromo-6-chloro-3-indoxyl phosphate, further, the colony formed bysuch strain turns magenta. When S. intermedius having both the abilityto produce α-glucosidase and the ability to produce phosphatase amongstaphylococcal bacteria other than S. aureus is present in the medium orthe culture layer of the microorganism culture substrate according tothe present aspect that contains the color developers in combinationdescribed above, in contrast, the strain grows, and the resulting colonyturns gray. When, among staphylococcal bacteria other than S. aureus,staphylococcal bacteria other than S. intermedius, such as S.saprophyticus subsp. saprophyticus, S. xylosus, or S. sciuri, is presentin the medium or the culture layer of the microorganism culturesubstrate, the growth of the strain is inhibited by colistin sodiummethanesulfonate at high concentration. Thus, the ability to produceα-glucosidase and/or the ability to produce phosphatase are notexpressed, and the color is not developed. When bacteria of Bacillusother than staphylococci are present in the medium or the culture layerof the microorganism culture substrate according to the present aspectthat contains the color developers in combination described above, thegrowth of the strain is inhibited, or the strain grows and the resultingcolony turns gray or blue. Therefore, S. aureus could be identified withhigh accuracy on the basis of the color of the formed microbial colony.

As described above, the medium and the microorganism culture substrateaccording to an aspect of the present invention enable selectivedetection of S. aureus without detecting staphylococcal bacteria otherthan S. aureus as false-positive strains. With the use of the medium andthe microorganism culture substrate according to an aspect of thepresent invention, accordingly, S. aureus in various targets can beselected rapidly with high accuracy.

EXAMPLES

The present invention is described in greater detail with reference tothe following examples. However, the technical scope of the presentinvention is not limited to these examples.

<I: Production of Microorganism Culture Sheet>

As a substrate sheet 10, a synthetic paper composed mainly of syntheticresin was prepared (YUPO®, Yupo Corporation). The configuration of thesubstrate sheet 10 was oblong. The thickness of the substrate sheet 10was 270 μm, the length of a longer side of the substrate sheet 10 was 90mm, and the length of a shorter side of the substrate sheet 10 was 72mm.

A circular frame layer 20 made of ethylene-vinyl acetate-based hot-meltresin was provided on an upper surface of the substrate sheet 10. Thewidth of the frame layer 20 (the gap between the outer periphery and theinner periphery of the frame layer 20) was 1 mm, the inner diameter ofthe frame layer 20 (a diameter of a circle defined by the innerperiphery) was 50 mm, and the height of the frame layer 20 was 750 μm.

A medium liquid for forming a culture layer 30 was applied in a regionsurrounded by the frame layer 20 on an upper surface of the substratesheet 10. The medium liquid comprises solid components, includingnutrient components, a color developer, a selection agent, a fixingagent, a gelling agent and a plasticizer, and a solvent for viscositymodification (methanol). As nutrient components, tryptone, soytone,yeast extract, sodium pyruvate, D(−)-mannitol, potassium phosphate, andglycine were used. Solid components included in the medium liquid areshown in Table 1.

TABLE 1 Components Experiment 1 Experiment 2 Experiment 3 Nutrientcomponent 56 56 56 Lithium chloride 16.8 16.8 16.8 Sodium azide 0.050.05 0.05 Nalidixic acid 0.019 0.019 0.019 6-Chloro-3-indoxyl-α-D- 0.83— 0.83 glucoside 5-Bromo-3-indoxyl phosphate — 0.83 0.83Polyvinylpyrrolidone 59 59 59 Psyllium seed gum 72 72 72 Guar gum 107107 107 Glycerin 23.5 23.5 23.5

The medium liquid applied to the substrate sheet 10 was dried to removemethanol via evaporation, and the culture layer 30 was formed on anupper surface of the substrate sheet 10. A thickness of the culturelayer 30 was 200 μm. The thickness of the culture layer 30 wasdetermined by measuring the distance between the lower surface and theupper surface of the substrate sheet 10 and the distance between thelower surface of the substrate sheet 10 and the upper surface of thedried culture layer 30 at 5 positions and calculating the average ofdifferences in both distances.

On the upper surface of the substrate sheet 10, a double-sided tape witha length of 6 mm and a width of 72 mm was applied as a fixing member 25along a shorter side of the substrate sheet 10. Subsequently, a coverfilm 40 having a substrate layer, a printed layer, and a water repellentlayer was prepared. The substrate layer was formed of transparentoriented polypropylene with a thickness of 40 μm. On the printed layer,lines defining squares with intervals of 10 mm were provided. The waterrepellent layer was formed of resin mainly composed of polyamide. Thelength of a longer side of the cover film 40 was 95 mm, and the lengthof a shorter side thereof was 72 mm. Subsequently, a region in thevicinity of a shorter side of the cover film 40 was bonded to thesubstrate sheet 10 via the fixing member 25. Thus, the microorganismculture sheet was produced.

<II: Detection of Microorganism>

Experiment 1: Test for Detecting Microorganisms Using a MediumContaining a Color Developer that Develops Color in the Presence ofα-Glucosidase

A given type of microbial strains to be tested were inoculated into aliquid medium (SCD broth medium), and culture was conducted at 35° C.for 24 hours to prepare a test microbial suspension. This test microbialsuspension was diluted with phosphate buffered physiological saline toadjust the cell density to about 1 to 500 cfu/ml. The resulting testmicrobial sample (1 ml) was inoculated into the culture layer of themicroorganism culture sheet prepared with the use of the medium liquidcontaining the components shown in Table 1 using a micropipette. Themicroorganism culture sheet into which the sample had been inoculatedwas allowed to stand in an incubator and culture was then conducted at35° C. for 24 or 48 hours. After the completion of culture, themicroorganism culture sheet was removed from the incubator. The color ofthe colony formed in the culture layer of the microorganism culturesheet and the color intensity were visually inspected. Each testmicrobial strain was subjected to the experiment described above. Thenames of the microbial strains to be tested used for the experiment andthe conditions and the color of the colonies formed by the strains areshown in Table 2.

TABLE 2 Culture for 24 hours Culture for 48 hours Colony Colony Strainformation Color formation Color Staphylococcus aureus Occurred PinkOccurred Pink ATCC25923 Staphylococcus aureus Occurred Pink OccurredPink NBRC100910 Staphylococcus aureus Occurred Pink Occurred PinkNBRC12732 Staphylococcus intermedius Occurred Pink Occurred PinkATCC29663 Staphylococcus hyicus Occurred No color Occurred PinkATCC11249 Staphylococcus epidermidis Occurred No color Occurred PinkNBRC12993 Staphylococcus saprophyticus Occurred Pink Occurred Pinksubsp. saprophyticus NBRC102446 Staphylococcus xylosus Occurred No colorOccurred Pink NBRC109770 Staphylococcus sciuri Occurred No colorOccurred Pink ATCC29062 Staphylococcus simulans Occurred Pink OccurredPink NBRC109714 Staphylococcus haemolyticus Occurred No color OccurredPink NBRC109768 Staphylococcus warneri Not occurred — Not occurred —NBRC109769 Staphylococcus hominis Not occurred — Occurred PinkATCC700586 Staphylococcus cohnii Not occurred — Occurred Pink subsp.cohnii NBRC109713 Staphylococcus capitis Not occurred — Not occurred —subsp. captis ATCC27840 Bacillus cereus Occurred Pink Occurred Pink(Strain derived from medium powder) Bacillus licheniformis Occurred PinkOccurred Pink NBRC12200 Bacillus subtilis Not occurred — Not occurred —NBRC3134

As shown in Table 1, the microorganism culture sheet prepared with theuse of the medium liquid containing the components of Experiment 1contains only 6-chloro-3-indoxyl-α-D-glucoside that develops color inthe presence of α-glucosidase as a color developer in the medium of theculture layer, but it does not contain colistin sodium methanesulfonateas a selection agent therein. For example, JP 2004-524041 A (PatentLiterature 2) describes that S. aureus can be distinguished from otherstaphylococcal bacteria and detected with the use of a culture mediumcontaining, as a color developer, an enzyme substrate that developscolor in the presence of α-glucosidase.

When the microorganism culture sheet of Experiment 1 was used, as shownin Table 2, colonies formed by several staphylococcal bacteria otherthan S. aureus turned pink. Also, colonies formed by 2 bacterial speciesof Bacillus turned pink. The results demonstrate that the staphylococcalbacteria other than S. aureus and the bacteria of Bacillus may bedetected as false-positive strains with the use of a microorganismculture sheet comprising a culture layer that contains, as a colordeveloper, only 6-chloro-3-indoxyl-α-D-glucoside, but does not contain,as a selection agent, colistin sodium methanesulfonate.

Experiment 2: Test for Detecting Microorganisms Using a MediumContaining a Color Developer that Develops Color in the Presence ofPhosphatase

A test microbial sample was prepared in the same manner as inExperiment 1. With the use of a micropipette, 1 ml of the test microbialsample was inoculated into a culture layer of the microorganism culturesheet prepared with the use of the medium liquid containing thecomponents shown in Table 1. In the same manner as with Experiment 1,the microorganism culture sheet into which the sample had beeninoculated was subjected to incubation, and the color of the colonyformed in the culture layer of the microorganism culture sheet and thecolor intensity were visually inspected. Each test microbial strain wassubjected to the experiment described above. The names of the microbialstrains to be tested used for the experiment and the conditions and thecolor of the colonies formed by the strains are shown in Table 3.

TABLE 3 Culture for 24 hours Colony Strain formation ColorStaphylococcus aureus Occurred Dark blue ATCC25923 Staphylococcus aureusOccurred Dark blue NBRC100910 Staphylococcus aureus Occurred Dark blueNBRC12732 Staphylococcus intermedius Occurred Dark blue ATCC29663Staphylococcus hyicus Occurred Light blue ATCC11249 Staphylococcusepidermidis Occurred Light blue NBRC12993 Staphylococcus saprophyticusOccurred No color subsp. saprophyticus NBRC102446 Staphylococcus xylosusOccurred Light blue NBRC109770 Staphylococcus sciuri Occurred Light blueATCC29062 Staphylococcus simulans Occurred No color NBRC109714Staphylococcus haemolyticus Occurred No color NBRC109768 Staphylococcuswarneri Not occurred — NBRC109769 Staphylococcus hominis Not occurred —ATCC700586 Staphylococcus cohnii Not occurred — subsp. cohnii NBRC109713Staphylococcus capitis Not occurred — subsp. captis ATCC27840 Bacilluscereus Occurred Dark blue (Strain derived from medium powder) Bacilluslicheniformis Occurred Dark blue NBRC12200 Bacillus subtilis Notoccurred — NBRC3134

As shown in Table 1, the microorganism culture sheet prepared with theuse of the medium liquid containing the components of Experiment 2contains only 5-bromo-3-indoxyl phosphate that develops color in thepresence of phosphatase as a color developer in the medium of theculture layer, but it does not contain colistin sodium methanesulfonateas a selection agent therein.

When the microorganism culture sheet of Experiment 2 was used, as shownin Table 3, colonies formed by several staphylococcal bacteria otherthan S. aureus turned dark blue to light blue. Also, colonies formed by2 bacterial species of Bacillus turned dark blue. The resultsdemonstrate that staphylococcal bacteria other than S. aureus andbacteria of Bacillus may be detected as false-positive strains with theuse of a microorganism culture sheet comprising a culture layer thatcontains, as a color developer, only 5-bromo-3-indoxyl phosphate, butdoes not contain, as a selection agent, colistin sodiummethanesulfonate.

Experiment 3: Test for Detecting Microorganisms Using a MediumContaining Two Types of Color Developers

A test microbial sample was prepared in the same manner as inExperiment 1. With the use of a micropipette, 1 ml of the test microbialsample was inoculated into a culture layer of the microorganism culturesheet prepared with the use of the medium liquid containing thecomponents shown in Table 1. In the same manner as with Experiment 1,the microorganism culture sheet into which the sample had beeninoculated was subjected to incubation, and the color of the colonyformed in the culture layer of the microorganism culture sheet and thecolor intensity were visually inspected. Each test microbial strain wassubjected to the experiment described above. The names of the microbialstrains to be tested used for the experiment and the conditions and thecolor of the colonies formed by the strains are shown in Table 4.

TABLE 4 Culture for 24 hours Colony Strain formation ColorStaphylococcus aureus Occurred Dark blue ATCC25923 Staphylococcus aureusOccurred Dark blue NBRC100910 Staphylococcus aureus Occurred Dark blueNBRC12732 Staphylococcus intermedius Occurred Purple ATCC29663Staphylococcus hyicus Occurred Pink ATCC11249 Staphylococcus epidermidisOccurred No color NBRC12993 Staphylococcus saprophyticus Occurred Pinksubsp. saprophyticus NBRC102446 Staphylococcus xylosus Occurred No colorNBRC109770 Staphylococcus sciuri Occurred Light blue ATCC29062Staphylococcus simulans Occurred Pink NBRC109714 Staphylococcushaemolyticus Occurred Pink NBRC109768 Staphylococcus warneri Notoccurred — NBRC109769 Staphylococcus hominis Not occurred — ATCC700586Staphylococcus cohnii Not occurred — subsp. cohnii NBRC109713Staphylococcus capitis Not occurred — subsp. captis ATCC27840 Bacilluscereus Occurred Pink (Strain derived from medium powder) Bacilluslicheniformis Occurred Pink NBRC12200 Bacillus subtilis Not occurred —NBRC3134

As shown in Table 1, the microorganism culture sheet prepared with theuse of the medium liquid containing the components of Experiment 3contains, as color developers, 6-chloro-3-indoxyl-α-D-glucoside thatdevelops color in the presence of α-glucosidase and 5-bromo-3-indoxylphosphate that develops color in the presence of phosphatase in themedium of the culture layer, but it does not contain colistin sodiummethanesulfonate as a selection agent therein.

When S. aureus is present in the medium in the culture layer of themicroorganism culture sheet of Experiment 3, the colony formed by suchstrain was deduced to turn, for example, purple, which is a mixed colorof pink developed by α-glucosidase activity and blue to dark bluedeveloped by phosphatase activity. When the microorganism culture sheetof Experiment 3 was used, however, the colony formed by S. aureussurprisingly turned dark blue, as shown in Table 4. In contrast,staphylococcal bacteria other than S. intermedius and S. sciuri did notform the colony, the colony formed thereby did not develop color, or thecolony turned pink. The colony formed by S. intermedius turned purple.Such patterns of color development are deduced to occur since S.intermedius expresses both α-glucosidase and phosphatase. Accordingly,S. aureus can be specifically detected by identifying a sample thatformed the dark blue colony as a positive strain, and also a sample thatdid not form the colony, a sample that formed the colony which did notdevelop color or a sample that formed the colony which turned pink as anegative strain. However, the colony of S. sciuri turned light blue.This indicates that such strain may be detected as a false-positivestrain.

Experiment 4: Test for Detecting Microorganisms Using a MediumContaining Colistin Sodium Methanesulfonate

A test microbial sample was prepared in the same manner as inExperiment 1. Also, the microorganism culture sheet was prepared in thesame manner as in Experiment 1. The medium liquid used for preparing themicroorganism culture sheet contains the solid components same as thoseused in Experiment 3 as shown in Table 1, a given amount of colistinsodium methanesulfonate at the final mass concentration as shown inTable 5, and a solvent for viscosity modification (methanol). With theuse of a micropipette, 1 ml of the test microbial sample was inoculatedinto a culture layer of the microorganism culture sheet. In the samemanner as with Experiment 1, the microorganism culture sheet into whichthe sample had been inoculated was subjected to incubation, culture wasconducted for 24 hours, and the color of the colony formed in theculture layer of the microorganism culture sheet and the color intensitywere visually inspected. Each test microbial strain was subjected to theexperiment described above. The names of the microbial strains to betested used for the experiment and the conditions of the colonies formedby the strains are shown in Table 5. In the table, a blank columnindicates that a colony was formed under normal growth condition, andthe term “Inhibited” indicates the condition under which the growth wasinhibited. The colistin sodium methanesulfonate concentration indicatedin the upper column is mass concentration (mg/cm³) relative to the totalvolume of the dried culture layer before the sample was inoculatedthereinto, and the concentration indicated in the lower column is massconcentration (mg/ml) relative to the total volume of the samplesolution that was inoculated into the culture layer at the time of use.

TABLE 5 Colistin sodium methanesulfonate (Upper column: mg/cm³; Lowercolumn: mg/ml) 0 0.3 0.5 0.8 1.0 1.3 1.5 1.8 2.0 2.3 Strain 0 0.1 0.20.3 0.4 0.5 0.6 0.7 0.8 0.9 Staphylococcus aureus ATCC25923Staphylococcus aureus NBRC100910 Staphylococcus aureus NBRC12732Staphylococcus intermedius ATCC29663 Staphylococcus saprophyticusInhibited Inhibited Inhibited Inhibited Inhibited Inhibited InhibitedInhibited subsp. saprophyticus NBRC102446 Staphylococcus xylosusInhibited NBRC109770 Staphylococcus sciuri Inhibited ATCC29062Staphylococcus carnosus Inhibited Inhibited Inhibited InhibitedInhibited (Derived from coast beef) Colistin sodium methanesulfonate(Upper column: mg/cm³; Lower column: mg/ml) 2.5 2.8 3.1 3.3 3.6 3.8 4.1Strain 1   1.1 1.2 1.3 1.4 1.5 1.6 Staphylococcus aureus ATCC25923Staphylococcus aureus NBRC100910 Staphylococcus aureus InhibitedInhibited Inhibited Inhibited Inhibited NBRC12732 Staphylococcusintermedius Inhibited Inhibited Inhibited Inhibited ATCC29663Staphylococcus saprophyticus Inhibited Inhibited Inhibited InhibitedInhibited Inhibited Inhibited subsp. saprophyticus NBRC102446Staphylococcus xylosus Inhibited Inhibited Inhibited Inhibited InhibitedInhibited Inhibited NBRC109770 Staphylococcus sciuri Inhibited InhibitedInhibited Inhibited Inhibited Inhibited Inhibited ATCC29062Staphylococcus carnosus Inhibited Inhibited Inhibited InhibitedInhibited Inhibited Inhibited (Derived from coast beef)

As shown in Tables 1 and 5, the microorganism culture sheet preparedwith the use of the medium liquid containing the components ofExperiment 4 contains, as color developers,6-chloro-3-indoxyl-α-D-glucoside that develops color in the presence ofα-glucosidase and 5-bromo-3-indoxyl phosphate that develops color in thepresence of phosphatase and, as a selection agent, colistin sodiummethanesulfonate at the concentration shown in Table 5 in the medium ofthe culture layer.

As shown in Table 5, colistin sodium methanesulfonate did not inhibitthe growth of the S. aureus ATCC25923 strain and the NBRC100910 strainat the tested concentration of 0.3 to 4.1 mg/cm³ (i.e., 0.1 to 1.6 mg/mlin the sample solution). Regarding the S. aureus NBRC12732 strain alone,the growth thereof was inhibited with the use of the microorganismculture sheet containing colistin sodium methanesulfonate at 3.1 mg/cm³(i.e., 1.2 mg/ml in the sample solution). In contrast, the growth of S.saprophyticus was inhibited with the use of the microorganism culturesheet containing colistin sodium methanesulfonate at 0.5 mg/cm³ (i.e.,0.2 mg/ml in the sample solution) or more even though the strain was aGram-positive bacterium. Also, the growth of S. xylosus and S. sciuriwas inhibited with the use of the microorganism culture sheet containingcolistin sodium methanesulfonate at 2.3 mg/cm³ (i.e., 0.9 mg/ml in thesample solution) or more. The growth of S. carnosus was inhibited withthe use of the microorganism culture sheet containing colistin sodiummethanesulfonate at 1.3 mg/cm³ (i.e., 0.5 mg/ml in the sample solution)or more. The results described above demonstrate that high-concentrationcolistin sodium methanesulfonate can be used as a selection agentexerting inhibitory effects on particular staphylococcal bacteria.

Experiment 5: Test for Detecting Microorganisms Using a MediumContaining Two Types of Color Developers and Colistin SodiumMethanesulfonate at High Concentration (1)

A test microbial sample was prepared in the same manner as inExperiment 1. Also, the microorganism culture sheet was prepared in thesame manner as in Experiment 1. The medium liquid used for preparing themicroorganism culture sheet contains the solid components same as thoseused in Experiment 3 as shown in Table 1, a given amount of colistinsodium methanesulfonate at the final mass concentration of 2.5 mg/cm³(i.e., 1 mg/ml in the sample solution), and a solvent for viscositymodification (methanol). With the use of a micropipette, 1 ml of thetest microbial sample was inoculated into a culture layer of themicroorganism culture sheet. In the same manner as with Experiment 1,the microorganism culture sheet into which the sample had beeninoculated was subjected to incubation, and the color of the colonyformed in the culture layer of the microorganism culture sheet and thecolor intensity were visually inspected. Each test microbial strain wassubjected to the experiment described above. The names of the microbialstrains to be tested used for the experiment and the conditions and thecolor of the colonies formed by the strains are shown in Table 6. FIG. 7and FIG. 8 show the conditions of colony formation of each strain aftercolor development and the color thereof. In the figures, FIG. 7(a) showsthe S. aureus ATCC25923 strain, FIG. 7(b) shows the S. aureus NBRC100910strain, FIG. 7(c) shows the S. aureus NBRC12732 strain, FIG. 7(d) showsS. intermedius, FIG. 7(e) shows S. hyicus, and FIG. 7(f) shows S.epidermidis. FIG. 8(a) shows S. saprophyticus subsp. saprophyticus, FIG.8(b) shows S. xylosus, FIG. 8(c) shows S. sciuri, FIG. 8(d) showsBacillus cereus, FIG. 8(e) shows Bacillus licheniformis, and FIG. 8(f)shows Bacillus subtilis.

TABLE 6 Culture for 24 hours Colony Strain formation ColorStaphylococcus aureus Occurred Dark blue ATCC25923 Staphylococcus aureusOccurred Dark blue NBRC100910 Staphylococcus aureus Occurred Dark blueNBRC12732 Staphylococcus intermedius Occurred Purple ATCC29663Staphylococcus hyicus Not occurred — ATCC11249 Staphylococcusepidermidis Not occurred — NBRC12993 Staphylococcus saprophyticus Notoccurred — subsp. saprophyticus NBRC102446 Staphylococcus xylosus Notoccurred — NBRC109770 Staphylococcus sciuri Not occurred — ATCC29062Staphylococcus simulans Not occurred — NBRC109714 Staphylococcushaemolyticus Not occurred — NBRC109768 Staphylococcus warneri Notoccurred — NBRC109769 Staphylococcus hominis Not occurred — ATCC700586Staphylococcus cohnii Not occurred — subsp. cohnii NBRC109713Staphylococcus capitis Not occurred — subsp. captis ATCC27840 Bacilluscereus Occurred Pink (Strain derived from medium powder) Bacilluslicheniformis Occurred Pink NBRC12200 Bacillus subtilis Not occurred —NBRC3134

When the microorganism culture sheet of Experiment 5 was used, as shownin Table 6, the colony of S. aureus turned dark blue as with the case ofExperiment 3 (FIG. 7(a) to (c)). In contrast, staphylococcal bacteriaother than S. intermedius did not form the colony. In particular, thegrowth of S. sciuri, the colony formation of which was confirmed inExperiments 1 to 3, was inhibited with the use of the microorganismculture sheet containing colistin sodium methanesulfonate at 2.5 mg/cm³(i.e., 1 mg/ml in the sample solution), and the colony was not formed.The colony of S. intermedius turned purple (FIG. 7(d)). The colonies oftwo types of bacteria of Bacillus turned pink (FIGS. 8(d) and (e)).Accordingly, it was found that S. aureus could be specifically detectedwithout detecting a false-positive strain even in the presence of otherstaphylococcal bacteria and Bacillus bacteria in the sample byidentifying a sample that formed the dark blue colony as a positivestrain, and also a sample that did not form the colony or a sample thatformed the colony which turned purple or pink as a negative strain.

Experiment 6: Test for Detecting Microorganisms Using a MediumContaining Two Types of Color Developers and Colistin SodiumMethanesulfonate at High Concentration (2)

A test microbial sample was prepared in the same manner as inExperiment 1. Also, the microorganism culture sheet was prepared in thesame manner as in Experiment 1. The medium liquid used for preparing themicroorganism culture sheet contains solid components prepared byreplacing the color developer that develops color in the presence ofα-glucosidase and the color developer that develops color in thepresence of phosphatase in the solid components used in Experiment 3 asshown in Table 1 by 5-bromo-4-chloro-3-indoxyl-α-D-glucoside and5-bromo-6-chloro-3-indoxyl phosphate, respectively. In addition, themedium liquid contains a given amount of colistin sodiummethanesulfonate at the final mass concentration of 2.5 mg/cm³ (i.e., 1mg/ml in the sample solution) and a solvent for viscosity modification(methanol). With the use of a micropipette, 1 ml of the test microbialsample was inoculated into a culture layer of the microorganism culturesheet. In the same manner as with Experiment 1, the microorganismculture sheet into which the sample had been inoculated was subjected toincubation, and the color of the colony formed in the culture layer ofthe microorganism culture sheet and the color intensity were visuallyinspected. Each test microbial strain was subjected to the experimentdescribed above. The names of the microbial strains to be tested usedfor the experiment and the conditions and the color of the coloniesformed by the strains are shown in Table 7. FIG. 9 to FIG. 11 show theconditions of colony formation of each strain after color developmentand the color thereof. In the figures, FIG. 9(a) shows the S. aureusATCC25923 strain, FIG. 9(b) shows the S. aureus NBRC100910 strain, FIG.9(c) shows the S. aureus NBRC12732 strain, FIG. 9(d) shows S.intermedius, FIG. 9(e) shows S. hyicus, and FIG. 9(f) shows S.epidermidis, respectively. FIG. 10(a) shows S. saprophyticus subsp.saprophyticus, FIG. 10(b) shows S. xylosus, FIG. 10(c) shows S. sciuri,FIG. 10(d) shows S. simulans, FIG. 10(e) shows S. haemolyticus, and FIG.10(f) shows S. warneri. FIG. 11(a) shows S. hominis, FIG. 11(b) shows S.cohnii, FIG. 11(c) shows S. capitis, FIG. 11(d) shows Bacillus cereus,FIG. 11(e) shows Bacillus licheniformis, and FIG. 11(f) shows Bacillussubtilis, respectively.

TABLE 7 Culture for 24 hours Colony Strain formation ColorStaphylococcus aureus Occurred Magenta ATCC25923 Staphylococcus aureusOccurred Magenta NBRC100910 Staphylococcus aureus Occurred MagentaNBRC12732 Staphylococcus intermedius Occurred Gray ATCC29663Staphylococcus hyicus Not occurred — ATCC11249 Staphylococcusepidermidis Not occurred — NBRC12993 Staphylococcus saprophyticus Notoccurred — subsp. saprophyticus NBRC102446 Staphylococcus xylosus Notoccurred — NBRC109770 Staphylococcus sciuri Not occurred — ATCC29062Staphylococcus simulans Not occurred — NBRC109714 Staphylococcushaemolyticus Not occurred — NBRC109768 Staphylococcus warneri Notoccurred — NBRC109769 Staphylococcus hominis Not occurred — ATCC700586Staphylococcus cohnii Not occurred — subsp. cohnii NBRC109713Staphylococcus capitis Not occurred — subsp. captis ATCC27840 Bacilluscereus Occurred Blue (Strain derived from medium powder) Bacilluslicheniformis Occurred Gray NBRC12200 Bacillus subtilis Not occurred —NBRC3134

When the microorganism culture sheet of Experiment 6 was used, as shownin Table 7, the colony of S. aureus turned magenta (FIG. 9(a) to (c)).In contrast, staphylococcal bacteria other than S. intermedius did notform the colony. In particular, the growth of S. sciuri, the colonyformation of which had been confirmed in Experiments 1 to 3, wasinhibited with the use of the microorganism culture sheet containingcolistin sodium methanesulfonate at 2.5 mg/cm³ (i.e., 1 mg/ml in thesample solution), and the colony was not formed. The colony of S.intermedius turned gray (FIG. 9(d)). The colonies of two types ofbacteria of Bacillus turned blue or gray (FIGS. 11(d) and (e)).Accordingly, it was found that S. aureus could be specifically detectedwithout detecting a false-positive strain even in the presence of otherstaphylococcal bacteria and Bacillus bacteria in the sample byidentifying a sample that formed the magenta colony as a positivestrain, and also a sample that did not form the colony or a sample thatformed the colony which turned gray or blue as a negative strain.

This description includes part or all of the content as disclosed in thedescription and/or drawings of Japanese Patent Application Nos.2016-016378 and 2016-113278, which are priority documents of the presentapplication.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

DESCRIPTION OF NUMERAL REFERENCES

-   10: Substrate sheet-   20: Frame layer-   25: Fixing member-   30: Culture layer-   40: Cover sheet

The invention claimed is:
 1. A microorganism culture substrate fordetecting Staphylococcus aureus comprising: a substrate and a culturelayer provided on an upper surface of the substrate, wherein the culturelayer comprises a medium comprising one or more nutrient components, acolor developer that develops color in the presence of α-glucosidase, acolor developer that develops color in the presence of phosphatase, andcolistin sodium methanesulfonate at 0.5 mg/cm³ or more.
 2. Themicroorganism culture substrate according to claim 1, which furthercomprises a cover sheet to cover the other side of the culture layerthat is not covered by the substrate, wherein the substrate is a sheet,and wherein the culture layer further comprises polyvinylpyrrolidone andone or more gelling agents.
 3. A method for detecting Staphylococcusaureus comprising: adding a microorganism-containing sample to theculture layer of the microorganism culture substrate according to claim1; incubating the microorganism culture substrate with the added sample;and identifying the presence of Staphylococcus aureus based on thegrowth of colored colony or colonies in the culture layer, wherein thecolony or colonies are colored by at least one color developer in theculture layer.
 4. The method according to claim 3, wherein themicroorganism-containing sample is a liquid, and the colistin sodiummethanesulfonate is 0.2 mg/ml or more after the sample is added to theculture layer.
 5. A method for detecting Staphylococcus aureuscomprising: adding a microorganism-containing sample to a mediumcomprising one or more nutrient components, a color developer thatdevelops color in the presence of α-glucosidase, a color developer thatdevelops color in the presence of phosphatase, and colistin sodiummethanesulfonate at 0.5 mg/cm³ or more; incubating the medium with theadded sample; and identifying the presence of Staphylococcus aureusbased on the growth of colored colony or colonies in the medium, whereinthe colony or colonies are colored by at least one color developer inthe medium.
 6. The microorganism culture substrate according to claim 1,wherein the color developer that develops color in the presence ofα-glucosidase is 6-chloro-3-indoxyl-α-D-glucoside, and the colordeveloper that develops color in the presence of phosphatase is5-bromo-3-indoxyl phosphate.
 7. The microorganism culture substrateaccording to claim 1, wherein the color developer that develops color inthe presence of α-glucosidase is5-bromo-4-chloro-3-indoxyl-α-D-glucoside, and the color developer thatdevelops color in the presence of phosphatase is5-bromo-6-chloro-3-indoxyl phosphate.
 8. The microorganism culturesubstrate according to claim 1, wherein the colistin sodiummethanesulfonate is 0.5 to 4.1 mg/cm³.
 9. The microorganism culturesubstrate according to claim 8, wherein the colistin sodiummethanesulfonate is 0.5 to 2.8 mg/cm³.
 10. The microorganism culturesubstrate according to claim 9, wherein the colistin sodiummethanesulfonate is 2.3 to 2.8 mg/cm³.
 11. The method according to claim5, wherein the color developer that develops color in the presence ofα-glucosidase is 6-chloro-3-indoxyl-α-D-glucoside, and the colordeveloper that develops color in the presence of phosphatase is5-bromo-3-indoxyl phosphate.
 12. The method according to claim 5,wherein the color developer that develops color in the presence ofα-glucosidase is 5-bromo-4-chloro-3-indoxyl-α-D-glucoside, and the colordeveloper that develops color in the presence of phosphatase is5-bromo-6-chloro-3-indoxyl phosphate.
 13. The method according to claim5, wherein the colistin sodium methanesulfonate is 0.5 to 4.1 mg/cm³.14. The method according to claim 13, wherein the colistin sodiummethanesulfonate is 0.5 to 2.8 mg/cm³.
 15. The method according to claim14, wherein the colistin sodium methanesulfonate is 2.3 to 2.8 mg/cm³.